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Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein–lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 °C. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface., Title in the list of papers of Anna Adler's thesis: Regulation of innate immune system by fragmented heparin-conjugated lipids on lipid bilayer membranes

A number of clinical treatment modalities involve contact between blood and biomaterials: these include extracorporeal circuits such as hemodialysis, cardiopulmonary bypass, plasmapheresis, and intravascular treatments. Common side effects arising from these treatments are caused by activation of the cascade systems of the blood. Many of these side effects are mediated via the complement system, including thromboinflammatory reactions and rejection of implants. Depending on the composition of the materials, complement activation is triggered via all the activation pathways but is by far mostly driven by the alternative pathway amplification loop. On biomaterial surfaces the alternative pathway amplification is totally unregulated and leads under optimal conditions to deposition of complement fragments, mostly C3b, on the surface leading to a total masking of the underlying surface. In this review, we discuss the mechanism of the complement activation, clinical consequences of the activation, and potential strategies for therapeutic regulation of the activation, using hemodialysis as demonstrator.

Background Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes

PURPOSE: the pathophysiologic mechanisms explaining differences in clinical outcomes following COVID-19 are not completely described. This study aims to investigate antibody responses in critically ill patients with COVID-19 in relation to inflammation, organ failure and 30-day survival. METHODS: All patients with PCR-verified COVID-19 and gave consent, and who were admitted to a tertiary Intensive care unit (ICU) in Sweden during March-September 2020 were included. Demography, repeated blood samples and measures of organ function were collected. Analyses of anti-SARS-CoV-2 antibodies (IgM, IgA and IgG) in plasma were performed and correlated to patient outcome and biomarkers of inflammation and organ failure. RESULTS: A total of 115 patients (median age 62 years, 77% male) were included prospectively. All patients developed severe respiratory dysfunction, and 59% were treated with invasive ventilation. Thirty-day mortality was 22.6% for all included patients. Patients negative for any anti-SARS-CoV-2 antibody in plasma during ICU admission had higher 30-day mortality compared to patients positive for antibodies. Patients positive for IgM had more ICU-, ventilator-, renal replacement therapy- and vasoactive medication-free days. IgA antibody concentrations correlated negatively with both SAPS3 and maximal SOFA-score and IgM-levels correlated negatively with SAPS3. Patients with antibody levels below the detection limit had higher plasma levels of extracellular histones on day 1 and elevated levels of kidney and cardiac biomarkers, but showed no signs of increased inflammation, complement activation or cytokine release. After adjusting for age, positive IgM and IgG antibodies were still associated with increased 30-day survival, with odds ratio (OR) 7.1 (1.5-34.4) and 4.2 (1.1-15.7), respectively. CONCLUSION: In patients with severe COVID-19 requiring intensive care, a poor antibody response is associated with organ failure, systemic histone release and increased 30

Patients in the intensive care unit (ICU) often straddle the divide between life and death. Understanding the complex underlying pathomechanisms relevant to such situations may help intensivists select broadly acting treatment options that can improve the outcome for these patients. As one of the most important defense mechanisms of the innate immune system, the complement system plays a crucial role in a diverse spectrum of diseases that can necessitate ICU admission. Among others, myocardial infarction, acute lung injury/acute respiratory distress syndrome (ARDS), organ failure, and sepsis are characterized by an inadequate complement response, which can potentially be addressed via promising intervention options. Often, ICU monitoring and existing treatment options rely on massive intervention strategies to maintain the function of vital organs, and these approaches can further contribute to an unbalanced complement response. Artificial surfaces of extracorporeal organ support devices, transfusion of blood products, and the application of anticoagulants can all trigger or amplify undesired complement activation. It is, therefore, worth pursuing the evaluation of complement inhibition strategies in the setting of ICU treatment. Recently, clinical studies in COVID-19-related ARDS have shown promising effects of central inhibition at the level of C3 and paved the way for prospective investigation of this approach. In this review, we highlight the fundamental and often neglected role of complement in the ICU, with a special focus on targeted complement inhibition. We will also consider complement substitution therapies to temporarily counteract a disease/treatment-related complement consumption.

Purpose: the pathophysiologic mechanisms explaining differences in clinical outcomes following COVID-19 are not completely described. This study aims to investigate antibody responses in critically ill patients with COVID-19 in relation to inflammation, organ failure and 30-day survival. Methods: All patients with PCR-verified COVID-19 and gave consent, and who were admitted to a tertiary Intensive care unit (ICU) in Sweden during March¿September 2020 were included. Demography, repeated blood samples and measures of organ function were collected. Analyses of anti-SARS-CoV-2 antibodies (IgM, IgA and IgG) in plasma were performed and correlated to patient outcome and biomarkers of inflammation and organ failure. Results: A total of 115 patients (median age 62 years, 77% male) were included prospectively. All patients developed severe respiratory dysfunction, and 59% were treated with invasive ventilation. Thirty-day mortality was 22.6% for all included patients. Patients negative for any anti-SARS-CoV-2 antibody in plasma during ICU admission had higher 30-day mortality compared to patients positive for antibodies. Patients positive for IgM had more ICU-, ventilator-, renal replacement therapy- and vasoactive medication-free days. IgA antibody concentrations correlated negatively with both SAPS3 and maximal SOFA-score and IgM-levels correlated negatively with SAPS3. Patients with antibody levels below the detection limit had higher plasma levels of extracellular histones on day 1 and elevated levels of kidney and cardiac biomarkers, but showed no signs of increased inflammation, complement activation or cytokine release. After adjusting for age, positive IgM and IgG antibodies were still associated with increased 30-day survival, with odds ratio (OR) 7.1 (1.5¿34.4) and 4.2 (1.1¿15.7), respectively. Conclusion: In patients with severe COVID-19 requiring intensive care, a poor antibody response is associated with organ failure, systemic histone release and increased 30

OBJECTIVE: Complement components, including C4d, can be found on activated platelets, a process associated with vascular disease in SLE. We investigated whether platelet C4d (PC4d) adds additional value to traditional and known lupus-associated risk factors when identifying SLE patients with vascular disease. METHODS: This cross-sectional study included 308 well-characterized SLE patients and 308 matched general population controls. PC4d deposition was analysed using flow cytometry. Values >95% of controls were considered as PC4d positive (+). aPL were determined by Luminex, and the LA test was performed by DRVVT. History of vascular disease (composite and as separate outcomes) was defined at inclusion. RESULTS: SLE patients had increased PC4d deposition as compared with population controls (50 vs 5%, P < 0.0001). PC4d+ positively associated with any vascular events, and separately with venous and cerebrovascular events, and also with all investigated aPL profiles. The association for any vascular event remained statistically significant after adjustment for traditional and SLE-associated risk factors (odds ratio: 2.3, 95% CI: 1.3, 4.3, P = 0.008). Compared with patients negative for both PC4d and LA, patients with double positivity were more likely to have vascular disease (odds ratio: 12.3, 95% CI: 5.4, 29.3; attributable proportion due to interaction 0.8, 95% CI: 0.4, 1.1). CONCLUSION: PC4d+ is associated with vascular events in SLE, independently of traditional and SLE-associated risk factors. Concurrent presence of PC4d and LA seem to interact to further increase the odds for vascular events. Prospective studies should examine whether the aPL/PC4d combination can improve prediction of vascular events in SLE and/or APS.

BACKGROUND: Complement system aberrations are implicated in numerous pathological conditions. Techniques used in complement diagnostic include monitoring of individual proteins, activation products or function using different types of immunoassays. Most recent techniques include multiplex assays which enable simultaneous detection of multiple complement components or activation products in the same sample thereby saving both sample volume and time. MATERIALS AND METHODS: We have tested the performance of the commercially available Human Complement Magnetic Bead multiplex assay MILLIPLEX MAP H (EMD Millipore Corporation, Billerica, MA, USA) for simultaneous determination of C1q, C4, C3, factor B, properdin and factor H as well as MBL, C2, factor D, factor I, C5 and C9 using plasma from 68 healthy blood donors. RESULTS: The main observation was very low levels of C3 determined by the MILLIPLEX assay: median value 16.4 mg/L (range 7.7-57.4) compared to the present reference value of 670-1290 mg/L used in the clinic (i.e., 60-fold lower). Discrepancies (although not as pronounced as for C3) were also found for C1q, factor B, factor H, C2 and C9. CONCLUSION: The MILLIPEX assay is highly inaccurate regarding C3 and less reliable for several other analytes, and implies that the company should reconstruct their assay for C3 since the results are not on par with the standard of other techniques used today.

To assess if markers of complement activation are associated with disease activity, C1q, C3, C3a and sC5b-9 levels in plasma and cerebrospinal fluid (CSF) were determined in 41 patients with clinically isolated syndrome (CIS) or remitting multiple sclerosis (RRMS), in a prospective longitudinal four-year cohort study. C1q in CSF (CSF-C1q) was significantly higher in patients than in controls. Baseline CSF-C1q and CSF-C3a correlated with several neuroinflammatory markers and neurofilament light chain levels. Baseline CSF-C3a correlated with the number of T2 lesions at baseline and new T2 lesions during follow-up. Baseline CSF-C3a was also significantly higher in patients with (n = 21) than in patients without (n = 20) signs of disease activity according to the NEDA-3 concept during one year of follow-up (p ≤ .01) Study results support that complement activation is involved in MS pathophysiology and that CSF-C3a carries prognostic information., Funding agencies: Medical Research Council of Southeast Sweden (FORSS); NEURO Sweden; Swedish Research CouncilSwedish Research Council [K2013-61X-22310-01-4, 2016-2075-5.1]; ALF grants, Region Ostergotland; Linnaeus University

Background. Hepatocyte transplantation (Hctx) is a potentially attractive method for the treatment of acute liver failure and liver-based metabolic disorders. Unfortunately, the procedure is hampered by the instant blood-mediated inflammatory reaction (IBMIR), a thromboinflammatory response elicited by the vascular innate immune system, causing activation of the coagulation and complement systems and clearance of transplanted cells. Observations have also revealed platelets adhered to the surface of the hepatocytes (Hc). To establish Hctx as a clinical treatment, all factors that trigger IBMIR need to be identified and controlled. This work explores the expression of von Willebrand factor (VWF) on isolated Hc resulting in tethering of platelets. Methods. VWF on Hc was studied by flow cytometry, confocal microscopy, immunoblot, and real-time polymerase chain reaction. Interaction between Hc and platelets was studied in a Chandler loop model. Adhesion of platelets to the hepatocyte surface was demonstrated by flow cytometry and confocal microscopy. Results. Isolated Hc constitutively express VWF on their cell surface and mRNA for VWF was found in the cells. Hc and platelets, independently of coagulation formed complexes, were shown by antibody blocking studies to be dependent on hepatocyte-associated VWF and platelet-bound glycoprotein Ib alpha. Conclusions. VWF on isolated Hc causes, in contact with blood, adhesion of platelets, which thereby forms an ideal surface for coagulation. This phenomenon needs to be considered in hepatocyte-based reconstitution therapy and possibly even in other settings of cell transplantation.

C3 glomerulopathy (C3G) is a severe kidney disease, which is caused by defective regulation of the alternative complement pathway. Disease pathogenesis is heterogeneous and is caused by both autoimmune and genetic factors. Here we characterized IgG autoantibodies derived from 33 patients with autoimmune C3 glomerulopathy. Serum antibodies from all 33 patients as well as purified IgGs bound to the in vitro assembled C3-convertase. Noteworthy, two groups of antibodies were identified: group 1 with strong (12 patients) and group 2 with weak binding C3-convertase autoantibodies (22 patients). C3Nef, as evaluated in a standard C3Nef assay, was identified in serum from 19 patients, which included patients from group 1 as well as group 2. The C3-convertase binding profile was independent of C3Nef. Group 1 antibodies, but not the group 2 antibodies stabilized the C3-convertase, and protected the enzyme from dissociation by Factor H. Also, only group 1 antibodies induced C3a release. However, both group 1 and group 2 autoantibodies bound to the C5-convertase and induced C5a generation, which was inhibited by monoclonal anti-C5 antibody Eculizumab in vitro. In summary, group 1 antibodies are composed of C3Nef and C5Nef antibodies and likely over-activate the complement system, as seen in hemolytic assays. Group 2 antibodies show predominantly C5Nef like activities and stabilize the C5 but not the C3-convertase. Altogether, these different profiles not only reveal a heterogeneity of the autoimmune forms of C3G (MPGN), they also show that in diagnosis of C3G not all autoimmune forms are identified and thus more vigorous autoantibody testing should be performed.

Aberrations in complement system functions have been identified as either direct or indirect pathophysiological mechanisms in many diseases and pathological conditions, such as infections, autoimmune diseases, inflammation, malignancies, and allogeneic transplantation. Currently available techniques to study complement include quantification of (a) individual complement components, (b) complement activation products, and (c) molecular mechanisms/function. An emerging area of major interest in translational studies aims to study and monitor patients on complement regulatory drugs for efficacy as well as adverse events. This area is progressing rapidly with several anti-complement therapeutics under development, in clinical trials, or already in clinical use. In this review, we summarized the appropriate indications, techniques, and interpretations of basic complement analyses, exemplified by a number of clinical disorders.

Iron-oxide nanoparticles (NPs) generated by environmental events are likely to represent health problems. α-Fe2O3 NPs were synthesized, characterized and tested in a model for toxicity utilizing human whole blood without added anticoagulant. MALDI-TOF of the corona was performed and activation markers for plasma cascade systems (complement, contact and coagulation systems), platelet consumption and release of growth factors, MPO, and chemokine/cytokines from blood cells were analyzed. The coronas formed on the pristine α-Fe2O3 NPs contained contact system proteins and they induced massive activation of the contact (kinin/kallikrein) system, as well as thrombin generation, platelet activation, and release of two pro-angiogeneic growth factors: platelet-derived growth factor and vascular endothelial growth factor, whereas complement activation was unaffected. The α-Fe2O3 NPs exhibited a noticeable toxicity, with kinin/kallikrein activation, which may be associated with hypotension and long-term angiogenesis in vivo, with implications for cancer, arteriosclerosis and pulmonary disease.

BACKGROUND: The complement system (CS) plays a role in the pathogenesis of a number of ocular diseases, including diabetic retinopathy (DR), glaucoma, uveitis, and age-related macular degeneration (AMD). Given that many of the complex eye-related degenerative diseases have limited treatment opportunities, we aimed to mimic the in vivo retinal degenerative process by developing a relevant co-culture system. METHOD AND MATERIALS: The adult porcine retina was co-cultured with the spontaneously arising human retinal pigment epithelial cells-19 (ARPE-19). RESULTS: Inflammatory activity was found after culture and included migrating microglial cells, gliosis, cell death, and CS activation (demonstrated by a minor increase in the secreted anaphylotoxin C3a in co-culture). CS components, including C1q, C3, C4, soluble C5b-9, and the C5a receptor, were expressed in the retina and/or ARPE cells after culture. C1q, C3, and CS regulators such as C4 binding protein (C4BP), factor H (CFH), and factor I (CFI) were secreted after culture. DISCUSSION: Thus, our research indicates that this co-culturing system may be useful for investigations of the CS and its involvement in experimental neurodegenerative diseases., Funding Agencies:Carmen and Bertil Regners Foundation Crown Princess Margaretas Committee for the Blind Olle Engqvist Foundation Sven and Dagmar Salens Foundation Ögonfonden ('The Eye Foundation') FORSS (Medical Research Council of Southeast Sweden) Ffaculty of Medical Sciences at Orebro University Linnaeus University Swedish Medical Research Council (VR)